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Abstract

The 1st International Workshop on Xenotropic Murine Leukemia Virus-Related Retrovirus (XMRV),
co-sponsored by the National Institutes of Health, The Department of Health and Human
Services and Abbott Diagnostics, was convened on September 7/8, 2010 on the NIH campus,
Bethesda, MD. Attracting an international audience of over 200 participants, the 2-day
event combined a series of plenary talks with updates on different aspects of XMRV
research, addressing basic gammaretrovirus biology, host response, association of
XMRV with chronic fatigue syndrome and prostate cancer, assay development and epidemiology.
The current status of XMRV research, concerns among the scientific community and suggestions
for future actions are summarized in this meeting report.

Introduction

In 2006, Urisman et al. [1] described the identification and characterization of a novel gammaretrovirus, xenotropic
murine leukemia virus-related virus (XMRV), in a small number of prostate cancers.
Subsequent studies of Schlaberg et al. [2] suggested that XMRV might have a broader distribution, and was present in both prostate
cancer patients and benign controls. XMRV is very closely related to endogenous proviruses
found in inbred (laboratory) mice, some of which cause lymphoma and other diseases
in mice. Due to the lack of functional receptor Xpr1, this virus does not replicate in most inbred mice, but grows well in human prostate
cancer cell lines. Interest in XMRV has recently intensified following the work of
Lombardi et al. [3], who detected XMRV in chronic fatigue syndrome (CFS) patients in clusters of cases
in Nevada and Florida-South Carolina. Virus could be detected through both antibodies
in serum and proviral DNA in peripheral blood mononuclear cells (PBMCs), and could
easily be cultured from PBMCs and plasma. However, although these and related studies
demonstrated an association of XMRV infection with at least two human diseases, causality
was not established.

Despite the significant increase in XMRV-related publications over the last 24 months,
the research community has failed to reach consensus on the origin of this virus,
its causative (or passenger) role in disease pathology, and the extent to which it
is prevalent in the human population. On the contrary, the numbers of studies identifying
XMRV in humans [1-6] are presently outweighed by reports from laboratories throughout the world that have
failed to detect the virus [7-15] which have now added to an increasing sense of confusion. Central to this has been
the lack of standardized nucleic acid-based or serological methods for detecting viral
nucleic acid and antibodies, respectively, as well as "gold standard" reference samples
with which individual groups can judge the selectivity and sensitivity of their protocols.
The 1st International Workshop on XMRV was therefore convened at the National Institutes of
Health, Bethesda, MD on September 7/8, 2010, with a goal of providing a public forum
to discuss these and related issues, including increasing concerns regarding mouse
DNA contamination, methods of sample handling and storage, use of antiretrovirals
currently available for HIV therapy, and progress in developing standard PCR and serological
reagents. In his introductory remarks, NIH Director Dr. Francis Collins urged the
225 attendees to maintain a healthy skepticism on potential causative roles of XMRV,
indicating that a solution to this conundrum requires an interdisciplinary and synergistic
effort from researchers in both the prostate cancer and CFS arenas. This report summarizes
overviews and research findings presented during the 2-day International Workshop.

Gammaretrovirus Biology

J. Coffin (Tufts University School of Medicine, Boston, Massachusetts) opened the Workshop by
providing background information on XMRV and the endogenous viruses of mice, summarizing
the basic properties of endogenous retroviruses and the original studies with XMRV
before proceeding to examine in more detail proviruses in the genomes of mice and
their effects on their hosts. Experiments in his laboratory have characterized xenotropic,
polytropic and modified polytropic endogenous proviruses, their distribution across
the mouse genome, co-evolution with different species of mice, and relationship to
viruses associated with prostate cancer and chronic fatigue syndrome. Dr. Coffin's
concluding statements set the tone for subsequent discussions of the Workshop. Uppermost
in his concerns were (i), conflicting reports on association with diseases (ii), lack
of insight into potential pathogenic mechanisms (iii), assay sensitivity used for
detecting XMRV and related viruses (iv), the well documented infection of human cells
passaged through nude mice by xenotropic MLV possibly initiating further spread, and
(v) ubiquitous presence of mice and mouse products likely containing multiple MLV
sequences. The magnitude of the problem was illustrated by considering a swimming
pool into which a drop of mouse blood was introduced, after which every milliliter
of water would contain enough DNA to give a positive signal using the current ultrasensitive
PCR techniques.

S. Chow (University of California, Los Angeles, California) described studies of the incidence
of XMRV and related MLV in healthy donors and patients with prostate cancer in two
cohorts, one in the U.S. (UCLA) and the other in China (Second Affiliated Hospital,
Hangzhou, Zhejiang and Ningbo Blood Center, Ningbo, Zhejiang) using an RT-PCR approach
with three different primer sets. Individuals were considered positive if one out
of the three tests yielded consistent positives. Perhaps surprisingly, an equal frequency
of positives was seen in the patient and control groups, and there was a higher incidence
of XMRV or MLV-related virus sequences associated with increasing age. An association
with the RNase L R462Q mutation previously linked with prostate cancer was not confirmed.
Env primers yielded the most positive results; including examples of XMRV, xenotropic,
polytropic and modified polytropic sequences. No examples of the 24-nt deletion in
the gag leader region characteristic of XMRV were detected with fragments amplified using
gag primers. Dr Chow subsequently described experiments to identify XMRV-host cell junctions
in samples from CFS patients. Such junction fragments were only found in two XMRV-positive,
patient-derived cell lines [16,17]. The sample size of XMRV integration sites in tumors is currently insufficient to
detect common integration sites with which to assess the role of insertional mutagenesis
as an oncogenic mechanism during XMRV infection.

A. Wlodawer (National Cancer Institute (NCI), Frederick, Maryland) opened his presentation by
pointing out the contribution of drugs targeting HIV-1 protease to highly-active antiretroviral
therapy, and the crucial role of structure-based drug design in their development.
Dr. Wlodawer reported the 2Å crystal structure of XMRV protease, which is responsible
for processing protein polyprotein precursors during virus maturation. As with related
retroviral proteases, the XMRV protein forms a homodimer, but despite overall similarities,
the XMRV and HIV-1 proteins are quite different, particularly at the dimer interface.
Overall, the structure resembles an internal domain from a human ubiquitin receptor
protein (Ddi1) that may function proteolytically during regulated protein turnover
in the cell. Recombinant XMRV protease showed a tendency for self-digestion, an observation
that will presumably be used in the development of XMRV protease inhibitors. An account
of this study will appear in Nature Structural and Molecular Biology.

O. Cingoz (Tufts University, Boston, Massachusetts) described different approaches to identify
a possible source of XMRV in mice. Sequence comparisons were conducted to design a
pair of PCR primers, spanning (i), a unique 2-nt insertion in the viral LTR and (ii)
the 24-nt deletion of the gag leader, allowing detection of XMRV against a background of closely related MLVs. Screening
over 70 inbred laboratory and wild derived mice failed to identify an endogenous provirus
with the predicted fragment. However in silico analyses showed that one or more proviruses carrying the 24-nt deletion is present
in several mouse strains, an observation that was confirmed by single genome PCR amplification
and Southern blotting experiments using an oligonucleotide probe spanning the deletion.
This probe reacts with a single provirus in these strains whose similarity with XMRV
remains to be determined. Together these observations strengthen the argument for
a murine origin of XMRV with recombination or mutation providing the LTR specific
change. Dr. Cingoz concluded by describing a highly sensitive assay for detecting
mouse DNA contamination using primers directed against Intracisternal A-type particle
(IAP) sequences, which are present at an estimated 1000 copies per haploid genome
[18]. An assay with such sensitivity, possibly complementing one directed against mouse
mitochondrial DNA, would guard against contaminating DNA in future PCR studies designed
to detect XMRV and related viruses in human samples.

The concluding presentation of R. Molinaro (Emory University, Atlanta, Georgia) described a novel gene product encoded by XMRV,
translated from a doubly spliced env mRNA of 1.2 kb and comprising an 11kD portion of the C-terminal region of the Envelope
polyprotein. Expression studies with a GFP fusion protein revealed a punctate pattern
of fluorescence present in both nucleus and cytoplasm. These studies are consistent
with, but do not prove, a possible role for the novel protein in the export of unspliced
viral RNA from the nucleus of XMRV-infected cells.

Host Response

The Host Response session was opened by R. Silverman (Cleveland Clinic, Cleveland, Ohio), who discussed the linkage of hereditary prostate
cancer with mutations in the ribonuclease L (RNase L) gene and discovery of XMRV [1]. In 86 patients, the finding that 8/20 were homozygous for the QQ mutation in RNase
L suggested a strong correlation [1], confirmed in one study [4] but not in others [2,5]. Data were presented showing that RNase L inhibited XMRV replication in cell culture.
Electron microscopy identified an enveloped retrovirus similar to MLV. A rhesus macaque
study with Francois Villinger and collaborators at Abbott Diagnostics (to be described),
showed that XMRV trafficked to prostate epithelium within 6-7 days post-infection,
but was observed only in stromal cells after 291 days. Similarly, in human prostate
cancer tissues XMRV was observed only in a small number of stromal cells [1]. The XMRV DNA in macaque PBMC in vivo was mutated by APOBEC3, relating to the subsequent
talk by K. Bishop. The androgen receptor element in the XMRV LTR U3 region was shown
to be sensitive to dihydroxytestosterone (DHT) in vitro, and DHT stimulated virus replication in vitro [19]. Dr. Silverman concluded with a statement that a causal link of XMRV to any human
disease remains to be established.

In view of the increasing interest in cellular inhibitors of retroviral replication,
K. Bishop (National Institute for Medical Research, UK) provided an overview of restriction
factors and their impact on XMRV replication [20]. Human APOBEC3G, a cytidine deaminase, which potently inhibits HIV replication through
lethal G -> A hypermutation, and to a lesser extent the related APOBEC3F, were also
shown to inhibit XMRV replication. However, while the HIV accessory protein Vif counters
APOBEC-mediated deamination by targeting it for proteasomal degradation, XMRV lacks
the counterpart. How XMRV achieves such "resistance" is presently unclear. Tetherin
(CD317/Bst2), a Type II membrane protein that localizes to multiple membrane compartments,
crosslinks nascent virions to the plasma membrane, preventing release of a variety
of retroviruses, filoviruses, gammaherpesviruses and arenaviruses. XMRV was likewise
sensitive to human, simian and murine tetherins. While HIV-1 Vpu counters tetherin-mediate
XMRV restriction in HeLa cells, the absence of such an accessory protein in XMRV again
begs the question of how host restriction is bypassed. Also, XMRV env cannot counteract
tetherin. Finally, XMRV is not sensitive to restriction by the intrinsic immune factor
TRIM5alpha, which can mediate an early block to HIV-1 replication. However, XMRV is
restricted by the mouse specific restriction factors, Fv1n and Fv1b. Understanding how XMRV evades host restriction factors in the course of natural
infection is clearly an important issue if developing antiviral strategies becomes
a priority.

Although the XMRV field is in its infancy, E. Sparger (University of California at Davis, California) highlighted issues that must be better
understood when considering vaccine development. These included the mode(s) of transmission,
pathogenesis in the host and immune correlates for control of virus replication. Dr.
Sparger's comments were based on the success of vaccinating domestic cats against
feline leukemia virus (FeLV), a related gammaretrovirus identified in 1964. Common
features of FeLV and XMRV include their potential association with immune suppression,
disease of the central nervous system, and induction of cancer. Successful strategies
included whole inactivated virus, recombinant surface glycoprotein, subunit vaccines
and nonadjuvanted canarypox-vectored live vaccine (ALVAC), with efficacy rates of
44% - 100% reported. Reiterating the cautious note that pathogenesis and immune correlates
for XMRV must be thoroughly characterized in order to inform vaccine design, Dr. Sparger
concluded by suggesting that newer and more novel approaches (e.g. vector systems,
molecular adjuvants, inclusion of multiple modalities) should further increase the
likelihood of success.

With the goal of establishing an animal model to study XMRV dissemination, tissue
tropism and pathogenicity, F. Villinger (Emory University, Atlanta, Georgia) summarized a collaborative study in which XMRV-infected
rhesus macaques were followed for various periods of time post-infection and euthanized
during acute infection or during chronic infection 146 and 289 days post-inoculation
[21]. Animals were monitored for immune parameters and viral replication as well as extensive
tissue collections and in situ analyses performed at necropsy. Animals showed transient viremia and induction of
antibodies as well as infection of prostate, spleen, liver, lymph nodes, lung and
jejunum. No evidence of pathogenesis was observed during the 9-month follow-up, together
with antibody responses that rapidly declined after infection and mostly undetectable
cell mediated immune responses, suggesting limited antigenic stimulation. However,
detailed in situ analysis of various organs and tissues detected virus replication at various times
post-infection. Demonstration of XMRV replication in reproductive organs (prostate,
seminal gland, testis as well as vagina and cervix) suggested a potential for sexual
transmission. In cautioning that expansion of the model is urgently needed, this study
provided a valuable model of human XMRV infection to assess long-term chronic infection,
pathogenesis, immunity and for validating potential vaccines.

The use of Gairdner's Shrewmouse, Mus pahari, as a small animal model of XMRV infection was presented by Y. Ikeda (Mayo Clinic, Rochester, Minnesota). The susceptibility of Mus pahari cells to XMRV is due to their novel receptor as previously described by C. Kozak and
co-workers [22]. The Kozak laboratory also showed that no wild mouse is resistant to xenotropic virus
and several laboratory mouse strains are susceptible to X-MLVs [23,24]. The Ikeda laboratory showed that M. pahari fibroblasts support XMRV replication in vitro, while inoculated mice demonstrated high levels of neutralizing antibodies 2 weeks
post-infection. XMRV proviral DNA was found mainly in blood, spleen and brain, suggesting
the virus was lympho- and neuro-tropic in Mus pahari [25]. Despite some practical difficulties (including small litters, relatively small spleen
and a lack of inbred strains), the Mus pahari model showed promise.

To uncover additional determinants of virus entry and identify entry restrictions
that could modulate trans-species transmissions, C. Kozak (NIAID, Bethesda, Maryland) examined evolution of Xpr1 in rodent species and the co-evolution of Xpr1 and xenotropic/polytropic MLVs (X/P-MLVs) in Mus species, extending this analysis to non-rodent species. Ten distinct phenotypes were
identified, distinguished by resistance to different X/P-MLVs, of which four were
known Xpr1 variants in Mus and a novel fifth allele was identified in Mus molossinus and Mus musculus. The geographic and species distribution of the five functional Xpr1 variants in Mus and their evolutionary association with endogenous X/P-MLVs were described. Specific
residues important for mouse X/P-MLV entry were demonstrated by mutational analysis,
which also indicated that, while XMRV relies on X-MLV entry determinants, it uniquely
requires at least one additional residue. In demonstrating the highly polymorphic
nature of the Xpr1 receptor, Dr. Kozak emphasized that, although all mammals carry functional receptors,
these differ in their ability to allow entry of the various human or mouse derived
viruses, reflecting sequence substitutions or deletions in the two extracellular loops
that carry receptor determinants.

XMRV and Prostate Cancer

In his introductory presentation, E. Klein (Cleveland Clinic, Cleveland, Ohio) addressed four questions: 1) why is prostate cancer
important? 2) is prostate cancer an infectious disease? 3) what is the role of XMRV
in prostate cancer? 4) what are the implications? Risk factors for prostate cancer
include age, race, family history and genetic factors that remain largely undefined.
Infections account for several types of cancers, but it is unknown if infectious agents
contribute to prostate cancer. However, mutations in genes involved in the host response
to infections or in immunity (e.g., RNASEL, MSR1 and TLR4) are associated with prostate cancer in humans. The RNASEL (HPC1) association is seen in multiple affected family members [26]. RNase L R462Q has reduced enzyme activity and doubles the risk of prostate cancer
when homozygous [27]. XMRV was discovered in such men (QQ genotype) with prostate cancer [1]. Published confirmatory studies of XMRV in prostate cancer were described [2,4,28], although only one suggested correlation with the RNASEL QQ genotype [4]. Possible reasons for studies failing to detect such an association [12] are that XMRV may not be truly associated with human disease, technique differences
(e.g. PCR details and unrecognized sequence variations), and geographical distribution
of the virus. Pathways to viral oncogenesis include insertional mutagenesis, proinflammatory
effects, oncogenic viral proteins, immune suppression and altered epithelial/stromal
interactions. For instance, cancer associated fibroblasts (but not normal fibroblast)
cause initiated epithelial cells to form large tumors in mice. The implications of
XMRV in prostate cancer include a potential biomarker for aggressive tumors [2]. In this regard, XMRV RNA was detected in a subset of expressed prostate secretion
(EPS) specimens from prostate cancer patients. Dr. Klein closed by suggesting that
if XMRV is shown to be a cause of prostate cancer it could lead to a vaccine, such
as the HPV vaccine used to prevent cervical cancer.

I. Singh (University of Utah, Salt Lake City, Utah) reviewed her work on the role of XMRV in
prostate cancer [2] and of antiretroviral drugs on XMRV infections in cell culture [29]. Compelling reasons for studying XMRV included a large number of prostate cancer
deaths, and a causal role for XMRV could spur new methods for prevention, biomarkers
for disease, help in resolving difficult cases and antiretroviral therapy. Rabbit
antisera to XMRV propagated in human 293T cells was used in immunohistochemistry (IHC)
experiments to probe human prostate tumor tissue sections (23% of which were positive).
Infected cells were almost all of the malignant epithelial type, including clusters
of such cells. In contrast, qPCR showed 6% were XMRV positive. Differences between
the two methods were attributed to very low viral loads, sampling differences, and
varying proportions of XMRV-infected cells. XMRV was associated with higher grades
of prostate cancer, but not tumor stage or age at diagnosis. Since association between
XMRV detection and the RNASEL SNP for R462Q could not be verified, the entire population may be susceptible to XMRV
infection. Lessons learned include that very small amounts of virus are present, contamination
from mouse tissues can occur, different sections of the same tumor may have different
amounts of virus, and that XMRV detection by IHC does not work well in tissue microarrays.

J. Petros (Emory University, Atlanta, Georgia) described XMRV variations in prostate cancer
cases, pointing out that there are relatively few SNP variations between reported
XMRV sequences and only limited full-length XMRV genome sequences in the public domain.
Evidence of XMRV in prostate cancer cases was obtained by an immunoassay detecting
XMRV-neutralizing antibodies, PCR and fluorescence in situ hybridization; results from seven different prostate cancer patients were in concordance
by all three methods [4]. Whole XMRV provirus amplification from malignant prostate tissues yielded amplicons
larger than 9 kb in contrast to the full-length 8.2 kb genome. The "extra" DNA has
not yet been identified, but smaller provirus amplicons were also found, indicating
both internal deletions and extensions. Dr. Petros suggested that aberrant XMRV integration
events and internal deletions result in substantial variation among integrated XMRV
sequences in prostate cancer tissues.

In contrast, K. Sfanos (Johns Hopkins University, Baltimore, Maryland) and co-workers failed to detect XMRV
in prostate cancer and benign tissue, pointing out no virus has been causally linked
to prostate cancer despite 30 years of searching. A real-time duplex PCR assay developed
in collaboration with A. Rein, NCI, Frederick, Maryland, was described. Both XMRV
and CCR5 (a single copy nuclear gene) were amplified in the same PCR well, the latter
confirming the quality of the DNA. As a positive control, CWR22Rv1 (an XMRV-infected
prostate cancer cell line) genomic DNA was diluted into HeLa or 293T cell genomic
DNA. The assay could detect ~20 copies of XMRV DNA in a vast excess of uninfected
cell DNA. DNA from 161 prostate tumors was assayed and, while CCR5 DNA was detected,
no XMRV-specific amplicon was obtained. IHC performed with polyclonal antisera against
MoMLV p30 Capsid (CA) and gp70 Envelope surface subunit (SU) protein likewise failed
to demonstrate staining of prostate tissues (596 prostate tumors and 452 benign prostate)
with either antiserum. Possible reasons for the negative results were that RNASEL R462Q homozygotes were not selected (a finding that is inconsistent between all of
the studies), that XMRV was not detected because of sequence variations, or that infected
cells are present at an extremely low level and below the limits of sensitivity. Differences
in PCR and serological methods between the different studies could also contribute
to the different findings [7].

N. Fischer (University Medical Center, Hamburg, Germany) presented on the prevalence of XMRV
in prostate cancer and viral mechanisms in tumorigenesis. Using RT-PCR of cryo-preserved
and fresh prostate tissues, XMRV was found only rarely in sporadic prostate cancer
(1/300) and in 1/70 benign controls [30]. Additionally, in collaboration with researchers at the Robert Koch Institute, Berlin,
Germany, only 1/50 benign prostatic hyperplasia cases was positive using polyclonal
antisera, while none of ten high grade prostate cancer cases was positive. To investigate
a possible indirect mechanism of carcinogenesis involving stromal cell infections,
studies with a cytokine antibody array indicated that several proteins were down-regulated
in prostate stromal fibroblasts (PrSc), including TIMP1&2, IGFBP2&4, HGF, and IL-13.
In contrast Gro-α was up-regulated. Interestingly, XMRV replication enhanced the migration
of LNCaP cells through Matrigel. Dr. Fischer suggested that XMRV could indirectly
contribute to prostate cancer through infection of stromal cells that release cytokines
affecting cell invasion and tumor progression.

B. Danielson (Baylor College of Medicine, Houston, Texas) sought to further define the geographic
distribution of XMRV among prostate cancer patients in the US by investigating the
association with RNASEL R462Q, and searching for correlations with clinical/pathological parameters [5]. The study involved 144 prostate cancer patients from Texas, with no preoperative
treatment, who underwent radical prostatectomy. Of these, 32 (22.2%) were determined
to be positive for XMRV. Nested PCR was used to amplify a 650 bp region of the env gene, and specimens were considered positive if one or more of three PCR replicates
yielded a correctly-sized amplicon. PCR products from 17 XMRV positive samples were
sequenced and found to be 98.6-100% identical to XMRV VP62. XMRV DNA was detected
in both normal and tumor tissues, and a correlation with the RNASEL QQ genotype could not be established. In addition, there was no correlation between
the presence of XMRV and tumor grade. Among factors important for the detection of
XMRV were the level of input DNA (650 ng) and amplification of the env gene (compared with gag and pol).

Additional talks summarizing prostate cancer studies included a presentation of F. Ruscetti (NCI, Frederick, Maryland). Antibody to XMRV Envelope protein was detected in plasma
from prostate cancer patients and expressed prostate secretions (EPS). Transmission
of XMRV from prostate cancer plasma and EPS to LNCaP cells in culture was demonstrated
immunologically by western blotting. Transmission of XMRV from plasma from NIH prostate
cancer patients to LNCaP cells was also shown by virus culture. Infectious virus and
antibodies against XMRV were observed in the blood of some prostate cancer patients.
Finally, virus was detected in prostate cancer plasma using a novel indicator cell
line developed at the NCI (see description of K. Lee's presentation below).

W. Switzer (CDC, Atlanta, Georgia) reported on 162 prostate cancer patients collected at Fox
Chase Cancer Center in Philadelphia, Pennsylvania. Using nested PCR on prostate tumor
tissue DNA, PCR products were obtained for gag, pol and env from one patient, from pol and env for a second, and pol alone from a third (all samples were negative for mouse mitochondrial DNA). However,
PCR was not successful in all replicates on individual samples (the range of XMRV-positive
to total numbers of replicates was between 1/4 to 7/9). There was 4.8 to 6.5% divergence
in a 167 bp pol sequence between the newly detected viruses and XMRV strains in the public databases,
and less than 2% divergence in a 323 bp gag sequence. All 162 plasma samples were antibody negative using western blot testing.
He also presented negative data on CFS and matched health controls that were previously
published [15].

J. Blomberg (Uppsala University, Uppsala, Sweden) assayed DNA by qPCR from trans-urethral resections
from prostate tissue of 400 patients with benign or malignant prostatic hyperplasia
from Umeå University Hospital, all of which were negative. There were three posters
on prostate cancer. N. Makarova (Emory University, Atlanta, Georgia) described an XMRV neutralizing antibody (NAb)
assay. Sera from 16 of 258 prostate cancer patients screened (6.2%) were positive
for XMRV Nab, which is lower than in their original study [4]. Y. Ikeda (Mayo Clinic, Rochester, Minnesota) showed a number of XMRV-positive biopsy samples
using nested PCR for gag (1 of 40 normal/benign, 4 of 70 intermediate prostate cancers, and 1 of 40 high-grade
prostate cancers at the Mayo Clinic). However, no XMRV-specific 24 bp deletion was
found in the gag leader regions of the PCR-positive clinical samples. J. Das Gupta (Cleveland Clinic, Cleveland, Ohio) described a novel qPCR assay for detecting XMRV
RNA in urine. About 26% of prostate cancer cases (31/120) were XMRV positive, while
1/22 urine specimens (4.3%) from normal healthy control individuals was XMRV positive.
Urine samples were negative for mouse mitochondrial DNA.

XMRV and Chronic Fatigue Syndrome

Pointing out that mouse cells contain ~50 copies each of endogenous MLV DNA, and that
less than one cell's worth could yield a detectable PCR product, B. Huber (Tufts University, Boston, Massachusetts) emphasized the urgent need to distinguish
contaminating mouse sequences from true XMRV infections. PBMC DNA was tested for XMRV
by qPCR and nested PCR, using primers specific for regions of the XMRV pol and gag genes, respectively. In addition Dr. Huber's group assessed potential mouse DNA contamination
using both qPCR for murine mitochondrial cytochrome oxidase and/or conventional PCR
for IAP DNA. While control experiments verified the sensitivity of all assays, her
group failed to detect XMRV in 184 CFS patients and 25 healthy controls. However,
positive results were obtained with some samples using the gag nested PCR assays. DNA sequencing of the PCR products revealed sequences identical
to those described from prostate cancer and CFS patients, in addition to sequences
more closely related to endogenous MLVs. However all samples testing positive for
XMRV or MLV DNA were also positive for mouse IAP and mitochondrial DNA, using either
assay. The source of this apparent contamination is under investigation.

Contrasting data was subsequently presented by M. Hansen (Cornell University, Ithaca, New York), who summarized a blinded study ("10/10/10"
study) designed to determine whether XMRV could be detected in PBMCs from three small
groups of subjects from a single geographic area. Study subjects (10 per group) were
classified as severely ill with, or recovered from, CFS. A control group lacked a
CFS diagnosis at any time. XMRV RNA was evaluated by nested RT-PCR, using gag primers [1]. In addition, PCR with mouse mitochondrial DNA primers were used on all cDNA preparations
to exclude mouse cell contamination. Gag sequences similar to polytropic MLV were detected in 8 of the severely-ill CFS patients,
3 of those who had recovered, and one of the controls. In order to determine whether
infectious virus could be recovered, a subset of these blood samples was incubated
with LNCaP cells followed by serial passage over several weeks. PCR analysis revealed
that cultures exhibiting gag sequences corresponded to those inoculated with CFS patient plasma. Although a relatively
small study, the prevalence of virus in severe or recovered CFS patients (55%) relative
to the control group (10%) strengthened the findings of Lombardi et al. [3].

Supporting the results of the Cornell study, S.C. Lo, (FDA/CBER, Bethesda, Maryland) reviewed his previously published findings on the
presence of MLV-related virus gene sequences in both CFS patients and healthy controls
[31]. A unique feature of this study was that portions of the CFS blood samples had been
maintained in frozen storage at -80°C from the mid 1990s. Using nested PCR, MLV-like
gag gene sequences could be amplified from PBMC DNA in 32 of 37 patients meeting the accepted
diagnostic criteria for CFS (86.5%) compared with only 3 of 44 (6.8%) healthy volunteer
blood donors. This study also detected viral RNA in the frozen plasma samples of these
CFS patients. However, gag and env sequences from CFS patients were more closely related to those of polytropic mouse
endogenous retroviruses than to XMRV. Recognizing the increasing concerns of contamination
(including the PCR primers themselves, laboratory reagents or commonly used viral
vectors), semi-nested PCR was used to demonstrate the absence of mouse mitochondrial
DNA. Dr. Lo pointed out in his concluding statements that additional studies are needed
to determine whether MLV-related viruses have a causal or secondary role in the development
of either CFS and prostate cancer.

Two European studies failed to detect XMRV infection in CFS and MS patients. A study
presented by N. Bannert (Robert Koch-Institute, Berlin, Germany) failed to detect the presence of antibodies
against gag and env in serum from 36 CFS patients (Fukada/CDC criteria), 50 multiple sclerosis patients
(fatigue severity scale 4,7+/-1.07) and 17 healthy individuals. In addition XMRV was
not detected in DNA isolated from stimulated PBMCs of 39 CFS, 50 MS and 30 healthy
controls using a nested PCR, and reverse transcriptase activity was absent from the
supernatant from stimulated PBMCs. Co-cultivation of PBMCs from a subset of patients
with LNCaP indicator failed to recover infectious virus.

J. Blomberg (Uppsala University, Uppsala, Sweden) investigated 50 CFS patients (Fukada criteria)
using virus isolation with LNCaP cells with patient plasma as inoculum from 40 of
these patients. Cultures were monitored at day 5 with integrase qPCR, potential positive cultures were passed for another 5 days. Though three cultures
were initially positive with a few copies, only one could be passed twice, but not
further. The other two initially positive cultures lost signal after the first passage.
Virus was not recovered. Serological testing was performed on 60 CFS samples and 100
blood donors using a multi-epitope approach with 22 peptides spanning Gag and Envelope
coated on Luminex beads. Peptides were designed to react broadly by conserved sequence
selection and inclusion of degenerate amino acids. Sera with a reaction above background
(non-coated beads) against minimally three peptides were considered positive. One
blood donor sample and two CFS samples reacted in this fashion. The authors concluded
that XMRV and related viruses are rare in Sweden.

A poster of Blanco et al. (Irsi Caixa Foundation, Barcelona, Spain) used an alternative approach to look for
XMRV. PBMCs from patients were immortalised by infection with Epstein Barr virus,
DNA extracted from cell pellets and tested for XMRV using real time PCR for pol (50-nt) and two nested PCR assays for gag and env genes. Eleven CFS patients (Fukada and Canadian criteria) and 5 healthy donors were
tested. Three CFS patients and one control were found positive in the nested env approach, one CFS patient and one control in the gag nested PCR, and four CFS patients and two controls in the real time pol PCR assays. Sequencing of the three gag amplicons found the 24-nt deletion characteristic of XMRV. The authors concluded that
EBV-transformed cell lines can harbour XMRV specific sequences.

The final presentation of the CFS session was delivered by J. Mikovits (Whittemore Peterson Institute, Reno, NV) who shared data on a recent study detecting
XMRV in the peripheral blood of CFS patients in the United Kingdom. All study patients
(50) met the requirements for CFS based on rigorous criteria. Peripheral blood from
these patients was shipped to NCI-Frederick for plasma and PBMC isolation, after which
serology and virus isolation were performed at two different laboratories. A multi-faceted
approach involved (i), nested RT-PCR for gag or env sequences (ii) detection of Env antibodies in plasma (iii), Western analysis from
LNCaP cells co-cultured with subject's cell-free plasma and (iv), immunological detection
of viral proteins expressed by activated PBMCs. Collectively, this study indicated
the presence of infectious virus in >60% of CFS patients. XMRV could be transmitted
either cell-associated or cell-free from both activated lymphocytes and plasma from
infected individuals by passage to LNCaP. Maintaining that the worldwide distribution
of XMRV is greater than previously assumed, Dr. Mikovits concluded her presentation
by calling for additional studies addressing the replication and pathogenesis of XMRV
in the human population, as well as prioritizing the development of antiviral agents
for testing in the appropriate clinical setting.

Development of XMRV Diagnostic Tools

A central theme of the Workshop was the availability to the research community of
reliable diagnostic reagents for nucleic acid testing, serology and virus culture.
Additionally, there was general consensus among attendees for including sensitive
PCR methods to detect contaminating MLV-related and mouse DNA. Based on the ability
to recapitulate a non-human primate model of XMRV infection [21], X. Qiu (Abbott Diagnostics, Illinois) presented an update on their collaboration with researchers
at Emory University and the Cleveland Clinic to develop a series of high-throughput
immunoassays for future epidemiological studies. Using serum from XMRV-inoculated
rhesus macaques and a viral lysate as source of antigen, antibody responses were detected
for surface subunit (SU) Envelope protein, 9 days post-inoculation, followed by the
trans-membrane protein p15E (TM) at Day 11 and Capsid (CA) at Day 14. Chemiluminescence-based
indirect (anti-human) and direct (double antigen) assay formats based on each of these
antigens are currently under development. By changing the source of SU from a bacterial
to a mammalian expression system and incorporating Avidin Biotin Complex signal amplification,
sensitivity of the SU immunoassay was improved >1000-fold. The prototype, direct SU
assay provided substantial discrimination between a blood donor negative population
and the 29 XMRV seropositive primate bleeds.

R. Bagni (SAIC-Frederick, Frederick, Maryland) summarized current NCI efforts to develop serological
reagents for XMRV. In the absence of a bona fide, pedigreed antibody-positive clinical control, a "training set" of 116 samples, 39
of which were designated XMRV-positive from the Lombardi et al. study, were examined for the presence of XMRV-specific antibodies. Of the 9 candidate
XMRV proteins, a strong serological response to the SU and TM, and CA was observed,
while to a lesser extent, antibodies to p12, MA and NC could be detected. Dr. Bagni
also introduced the concept of a "positivity algorithm", i.e. the number of XMRV antigens
for which an immunological response must be detected before designating a sample positive.
A total of 64 expression clones constructed for development of the NCI XMRV ELISA
has now been deposited at the NIH AIDS Research and Reference Reagent Program https://www.aidsreagent.orgwebcite to be accessed by researchers of the extramural community.

As a valuable complement of nucleic acid and serological assay reagents, K. Lee (NCI, Frederick, Maryland) described the development of a novel cell line to rapidly
assess XMRV or XMLV replication.

D

etectors of

E

xogenous

R

etroviral

S

equence

E

lements, or DERSE indicator cells, exploit a specialized retroviral vector containing
an inverted, intron-interrupted green fluorescent protein (GFP) reporter cassette.
Although GFP is not expressed within a target cell after an initial infection, transfer
of an intron-less vector to new cells during a second round of XMRV infection permitted
GFP expression, which can be easily monitored by microscopy. Importantly, the DERSE
cell line permits virus detection in a little as three days, representing a considerable
time saving over standard methods. Dr. Lee indicated that this cell line will be deposited
in the NIH AIDS Reagent Repository for use by researchers in the extramural community.
While clearly not intended for first-line (high throughput) analysis, the DERSE cell
line will most certainly find use as a confirmatory strategy.

M. Kearney (NCI, Frederick, Maryland) presented two highly-sensitive single-copy assays for detection
of both XMRV and MLV-related viruses in blood products. The first of these, the XMRV
single copy assay, or X-SCA, is a qualitative PCR assay (analogous to that employed
for HIV detection) capable of detecting a single pelletable virion in plasma or XMRV
DNA in whole blood or PBMC. As a complement, the XMRV single genome sequencing assay
(X-SGS) likewise facilitates individual sequencing of large genomic fragments. Preliminary
data indicated that X-SCA compared favorably in specificity and sensitivity with related
protocols under development at the FDA, CDC, Whittemore-Peterson Institute and Blood
Systems Research Institute. In combination, X-SCA and X-SGS are capable of discriminating
between XMRV and contaminating mouse endogenous viruses with 100% accuracy.

The concluding presentation of G. Simmons (Blood Systems Research Institute, San Francisco, California) set the stage for discussing
future actions to help resolve disparate results presented during the Workshop. The
Blood XMRV Scientific Research Working Group (Blood XMRV SRWG) was established as
a National Heart, Lung and Blood Institute (NHBLI) coordinated working group to design
and coordinate collaborative studies to standardize existing assays and investigate
the prevalence of XMRV in blood donors. A four-phase, multi-center study was described,
wherein Phase 1 involved PCR testing, in a blinded fashion, of analytical performance
panels comprising pedigreed negative blood and plasma spiked with serial dilutions
of XMRV infected cells and supernatants, respectively. In general, there was good
agreement between participating laboratories. Phase II will compare XMRV nucleic acid
detection in frozen PBMCs, whole blood and plasma from CFS patients previously identified
as viremic. Importantly, replicate blood specimens would be processed at different
storage intervals to determine whether the 2-4 day processing period common to many
blood donor repositories affects assay performance. Phases III and IV will extend
these studies to begin to examine the prevalence of XMRV in blood donors by both nucleic
acid and serological methods.

The Path Forward - Consensus and Caution

Considering the discrepancies between the different studies regarding the prevalence
of XMRV, it became abundantly clear that reaching consensus on protocols for PCR amplification,
for discriminating between XMRV and contaminating mouse endogenous viruses, and sample
storage and processing should be an immediate priority among groups studying XMRV.
The studies of the Blood XMRV SRWG are likely to be of great importance in developing
such a consensus. The availability of analytical performance panels comprising pedigreed
samples would also be an enormous benefit to researchers. The scientific community
might also consider establishing a "repository" where protocols can be publicly deposited
and compared, which could reveal nuances underlying the discrepancies observed when
similar reagents are used by different groups.

Finally, there was vigorous discussion about the use and timing of interventions targeted
against XMRV in CFS and prostate cancer patients. Although a small number of workshop
participants advocated the immediate use of antiretrovirals that have successfully
controlled HIV infection, and while a well-controlled, randomized clinical trial should
not be ruled out, proceeding with caution was emphasized. Until (a) a causal role
for XMRV in CFS or prostate cancer is firmly established (b), objective biomarkers
are defined, and (c) uniformly-accepted assays to monitor effects on virus replication
are in place, the off-label use of antiretrovirals and anecdotal reports of their
efficacy will be unacceptable to third party payers/regulators, and could potentially
keep valuable therapies out of reach of many patients.